# High capacity clinical SARS-CoV-2 molecular testing using combinatorial pooling

## TL;DR

A new single-stage pooled testing method called P-BEST was developed and used in Israel to test millions of SARS-CoV-2 samples efficiently during the pandemic.

## Contribution

P-BEST enables high-capacity clinical testing with a single-stage pooling strategy, validated for use at high positivity rates.

## Key findings

- P-BEST was clinically validated with 3636 side-by-side tests and detected all positive samples accurately.
- P-BEST reduced the number of PCR tests needed by 3.1-fold during the Alpha and Delta waves in Israel.
- A tablet-based solution enabled manual single-stage pooling in settings without automated equipment.

## Abstract

The SARS-CoV-2 pandemic led to unprecedented testing demands, causing major testing delays globally. One strategy used for increasing testing capacity was pooled-testing, using a two-stage technique first introduced during WWII. However, such traditional pooled testing was used in practice only when positivity rates were below 2%.

Here we report the development, validation and clinical application of P-BEST - a single-stage pooled-testing strategy that was approved for clinical use in Israel.

P-BEST is clinically validated using 3636 side-by-side tests and is able to correctly detect all positive samples and accurately estimate their Ct value. Following regulatory approval by the Israeli Ministry of Health, P-BEST was used in 2021 to clinically test 837,138 samples using 270,095 PCR tests - a 3.1fold reduction in the number of tests. This period includes the Alpha and Delta waves, when positivity rates exceeded 10%, rendering traditional pooling non-practical. We also describe a tablet-based solution that allows performing manual single-stage pooling in settings where liquid dispensing robots are not available.

Our data provides a proof-of-concept for large-scale clinical implementation of single-stage pooled-testing for continuous surveillance of multiple pathogens with reduced test costs, and as an important tool for increasing testing efficiency during pandemic outbreaks.

Testing samples for SARS-CoV-2 is usually done on one sample at a time. However, the unprecedented demand for testing during the COVID-19 pandemic led to the adoption of pooled testing strategies, where samples are combined before being tested. This strategy requires two rounds: first, each pool of samples is tested, and then a second testing round is performed on individual samples from positive pools. We developed and implemented a pooling method for SARS-CoV-2 that requires a single round of testing, thus enabling the shorter turnaround times required during a pandemic. The method was approved for clinical use in Israel and was used to successfully test 837,138 clinical samples using fewer than a third of the tests usually required. Our study provides a blueprint for rapid implementation of efficient high-throughput testing in future pandemics.

Zismanov, Shalem, Margolin-Miller et al. present the validation and clinical rollout of P-BEST, a combinatorial single-stage pooling method used for mass testing during the SARS-CoV-2 pandemic in Israel. The analysis is a proof-of-concept for the feasibility of using combinatorial pooling solutions for mass testing in clinical settings.